Research on reproduction is essential for captive breeding of endangered carnivore species.

Assisted reproductive technology (ART) has great potential for conservation, but its successful application in captive breeding programmes of endangered species is often compromised by limited background on species' biology. Although carnivore species benefit from knowledge obtained in domesticated species (dogs, cats and ferrets), the focus of research is different. In pet animals, research in reproduction has mainly been focused on ovarian function and contraception, although substantial progress has also been made in the field of in vitro embryo production, transgenic embryos and cloning to aid relevant medical models. In endangered species, however, research should focus on characterizing reproductive traits (cyclicity and seasonality) to unravel species-specific endocrine principles of reproduction physiology. Based on this knowledge, it is crucial to enhance the ability to manipulate female reproductive cycles, especially those of embryo recipients. Furthermore, research conducted on molecular and cellular mechanisms of gamete and embryo development, as well as on cryopreservation protocols of gametes and embryos, is required for successful implementation of advanced ART to wild carnivores. This review will provide a summary on the state of the art with focus on ART contributing to conservation breeding of endangered carnivores.

Embryonic resorption in context to intragestational corpus luteum regression: a longitudinal ultrasonographic study in the European brown hare (Lepus europaeus PALLAS, 1778).

Embryonic resorption is frequently observed in polytocous mammals. Often it occurs as partial litter resorption affecting only single conceptuses of a whole litter. The aim of the study was to describe the incidence and morphology of embryonic resorption in the European brown hare (Lepus europaeus). In 154 pregnancies viable conceptuses, conceptuses undergoing resorption and CL of pregnancy were ultrasonographically monitored during the entire gestation period. Resorptions were classified into (1) "pre-implantation resorptions," (2) "peri-implantation resorptions," and (3) "post-implantation resorptions." The incidence of resorption in the pre-implantation period was 9%, in the peri-implantation period 9%, and in the post-implantation period 24%. Post-implantation resorptions were found up to late pregnancy stages when fetal development was already in progress. The highest daily incidence of resorption was on Day 8 of the 42-day pregnancy. In 91% of the cases, the regression of one CL was observed, while an embryo was undergoing resorption at the same time. The number of resorptions did not significantly differ from the number of CL in regression during gestation, suggesting an interesting one-resorption-to-one-regression relationship. The ultrasonographic appearance of the luteal regression during pregnancy was similar to the morphology characteristic for postpartal luteolysis.

Embryo retrieval after hormonal treatment to control ovarian function and non-surgical artificial insemination in African lions (Panthera leo).

Assisted reproduction technologies are essential for propagating endangered wild felids. Artificial insemination (AI) has been reported in several wild feline species, but pregnancy rates are low, partially owing to failures of current hormonal stimulation protocols. Therefore, this study describes the application of reliable methods to monitor ovarian activity and the development of an effective hormonal protocol to induce oestrus and ovulation in African lions. Application of porcine FSH and porcine LH was shown to be effective for inducing follicular growth and ovulation, and this regimen appeared to be superior to protocols described earlier in terms of ovulation and fertilization rates. Furthermore, non-surgical AI was performed successfully in lions, and uterine-stage embryos were collected and cryopreserved. African lions may serve as a valuable model to develop assisted reproduction for propagation of relic zoo populations in the critically endangered Asian lion or Barbary lion.

In vivo tissue sampling of embryonic resorption sites using ultrasound guided biopsy.

In the polytocous European brown hare (Lepus europaeus) more than 23% of all successful implantations undergo embryonic resorption. The objective of the study was to establish a minimally invasive ultrasound guided biopsy technique to collect embryonic resorption tissue in vivo. The sampled material was genetically analysed to determine paternity and the sex of the embryo. Female hares were either mated or artificially inseminated and pregnancy was confirmed by ultrasound on day six post ovulation. Subsequent embryonic development was ultrasonographically monitored on a regular basis to detect embryos undergoing resorption. Cell material of the resorption site was collected under ultrasonographic control via transabdominal biopsy of the placenta or aspiration of resorption fluid. To avoid breathing movements during the biopsy, the animals were intubated and a short apnoea was evoked by assisted ventilation. The presence of embryonic cells in the biopsy material was confirmed by microsatellite analysis in 11 of the fluid samples (n = 28) and six of the placental samples (n = 8). The lower success rate in the fluid samples was attributed to the abundance of maternal cells which was confirmed by the analysis of fluid sample smears. Male sex of the embryos undergoing resorption was detected by SRY analysis for ten of the fluid samples and for one of the placental samples. The two biopsy techniques did not have any negative impact on the prenatal development of the healthy siblings nor did it influence the future breeding performance of the females that were biopsied.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males.

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100x10(6) spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.

Gonadal activity in male and female captive fossas (Cryptoprocta ferox) during the mating season.

The fossa is an endangered, mongoose-like carnivore species endemic to Madagascar with a breeding season (BS) in the southern hemisphere spring. For the present study, faecal samples of captive fossas were collected for over 1 year for five males and four non-pregnant females, and for two pregnant animals during the reproductive period. The goal was to assess gonadal activity using non-invasive hormone measurements of faecal testosterone (T) and gestagen metabolites using assays previously established in our laboratory and further validated in this study. All study animals were housed in northern hemisphere zoos. In males, the seasonal T metabolite profile revealed a peak in spring (March). High performance liquid chromatography (HPLC) analysis was used to characterize the faecal hormone metabolite composition. The highest immunoreactivity was detected in the position of dihydrotestosterone, whereas native T was not detected. In the two pregnant females, gestagen metabolite concentrations increased 4-9 days after the initial matings and remained elevated throughout gestation with concentrations dramatically higher than those of the non-pregnant females during the BS. In these females, gestagen metabolite analyses did not reveal a seasonal pattern similar to the males. The HPLC-analysis revealed that the major proportion of immunoreactivity was associated with an unknown metabolite, whereas native progesterone was undetectable. The seasonal hormone pattern of male fossas gives proof of the reproductive seasonality of this species. The elevated 5alpha-pregnan-3beta-ol-20-one levels in pregnant animals allows for the characterization of pregnancy in the fossa based on analysis of faecal steroid metabolite concentrations.

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys.

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulating hormone (rhFSH) for 8 d. Animals (n=17) with > or =5 follicles (> or =3mm) in their ovaries on Day 9 of ovarian stimulation were deemed good responders, whereas those with a lesser response were poor responders (n=4). For these two groups, the mean (+/-SD) numbers of oocytes retrieved were 44.3+/-21.4 and 11.0+/-4.6, respectively. In retrospect, the mean diameters of the ovaries and of the largest follicles, the total number of detectable follicles (diameter >0.5mm), and serum estradiol concentrations gradually increased during the stimulation period in the good responders but did not increase in the poor responders. Comparing good and poor responders, the number of ovarian follicles >0.5mm already exhibited a difference (12.9+/-6.5 vs. 2.9+/-1.3, respectively, P<0.05) on Day 1 of stimulation. However, for other end points, differences were not significant until at least Day 5. Moreover, good responders yielded a fivefold higher blastocyst development rate than that of poor responders (P<0.01). In conclusion, the number of ovarian follicles detected with ultrasonography could be useful to predict the response to FSH stimulation in non-human primates.

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis).

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 degrees C for 4 to 6h during transport or 37 degrees C for 1 to 1.5h) on sort efficiency and sperm quality was investigated. Staining at 15 degrees C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 degrees C after transport (33%) and resulted in superior sperm integrity after staining (43.8+/-11.3% vs. 19.6+/-12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of alpha-amylase or collagenase (0.5 and 3IU per 100 microL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.

Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys.

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II, and III were given 35, 18, and 9 IU recombinant human follicle-stimulating hormone (rhFSH), respectively, twice daily for 8 d; Group IV was given 18 IU rhFSH twice daily until the appearance of maximal increase in sex skin during the breeding season; and Group V was treated identically to Group II but during the nonbreeding season. In addition, nine menarchial monkeys (Group VI) were treated identically to Group II. Menarchial monkeys yielded two- to fivefold the numbers of MII oocytes (24.1) and almost twice the development potential of in vitro-fertilized oocytes (blastocyst rate: 50.0%) compared with those of the other groups. Moreover, prepubertal monkeys in Group V had approximately double the numbers of MII oocytes and in Groups IV and V twice the development potential compared with those of Groups I and II, whereas Group III did not respond to stimulation. The most prominent sex skin swelling was in association with peak serum estradiol concentrations, and good responses to stimulation were associated with reduced body temperatures. All stimulated monkeys had normal reproductive performance at adulthood, except those in Group I. In conclusion, gonadotropin stimulation of menarchial monkeys could be appropriate for addressing the high cost and limited availability of rhesus monkeys in studying reproductive biology in primates.

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni).

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p<0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p<0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p<0.01, p<0.03, p<0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p<0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.

Germany/Australia index of sperm sex sortability in elephants and rhinoceros.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season.

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations, number of oocytes retrieved, and in vitro blastocyst development following IVF. One group underwent a single stimulation regimen with 18 IU rhFSH i.m., followed by 1000 IU hCG, and serum steroid concentrations and ovarian status were determined in the following three menses. Another group was stimulated as before and then allocated into three subgroups; each subgroup was re-stimulated once at the beginning of the ensuing first, second, or third menses. In the final experiment, one group was stimulated with 37.5 IU rhFSH, whereas another group received 18 IU rhFSH. In subsequent cycles, all were re-stimulated twice with 18 IU rhFSH at time intervals of two menstrual cycles (MCs). At the first menses after stimulation, serum progesterone concentrations were significantly higher and the ovaries larger than before stimulation. Monkeys that were re-stimulated at the first menses responded poorly; at the second menses, progesterone concentrations and ovarian size recovered, but the number of oocytes retrieved from re-stimulated monkeys was still significantly reduced. However, animals that were re-stimulated in two MCs later responded well (i.e., percentage of the animals responding, oocytes recovered, and potential for fertilization and blastocyst formation). In conclusion, rhesus monkeys were likely to have similar ovarian responses to repeated stimulation with the same regimen spaced at least two MCs apart.

Effects of rhFSH dose on ovarian follicular response, oocyte recovery and embryo development in rhesus monkeys.

The objective was to study the effects of dose of recombinant human follicular stimulating hormone (rhFSH) for ovarian stimulation in rhesus monkeys. Nineteen pubertal and 109 adult female rhesus monkeys were given 37.5, 18, or 9 IU of rhFSH twice-daily for 8 days (total of 600, 300, or 150 IU of rhFSH per cycle, respectively; designated Regimens 1, 2 and 3). Ovarian responses were assessed with ultrasonography, serum concentrations of E2 and FSH, and by in vitro developmental potential (following IVF) of retrieved oocytes. Regimen 1 had more monkeys with very large follicles (diameter>8 mm) than Regimen 2 (P<0.05), which impaired development potential. However, there were no differences between Regimens 1 and 2 in oocyte recovery, whereas Regimen 3 did not elicit superovulation. The developmental potential of embryos obtained from Regimen 2 was higher than that of Regimen 1, as determined by culture to the blastocyst stage in vitro (proportion of blastocysts relative to collected MII oocytes was 55.8% versus 36.8% in pubertal and 63.8% versus 44.2% in adult monkeys; P<0.05 for each), and the results of embryo transfer from Regimen 2 were acceptable. In conclusion, we inferred that the optimal rhFSH dose for ovarian stimulation in rhesus monkeys was a total of 300 IU; this dose should be efficacious for ovarian stimulation as the quality or recovered oocytes was higher and the risk of overstimulation was reduced.

Monitoring testicular activity of male Eurasian (Lynx lynx) and Iberian (Lynx pardinus) lynx by fecal testosterone metabolite measurement.

The aim of the present study was to identify relevant fecal testosterone metabolites in the Eurasian lynx (Lynx lynx) using HPLC analysis and to evaluate the specificity of two testosterone immunoassays against these fecal metabolites. Finally, fecal hormone analysis was used to characterize seasonal reproductive activity of captive male Eurasian and Iberian (Lynx pardinus) lynx. Fecal samples from a male Eurasian lynx who received an i.v. injection of [3H]testosterone were subjected to HPLC analysis. All HPLC fractions were analyzed for radioactivity and androgen content by two testosterone immune assays (EIA and Testosterone-Immulite kits, DPC Biermann, Germany). Furthermore, fecal samples from four Eurasian lynx males (n=174) and three Iberian lynx (n=52) were collected throughout the year and fecal testosterone metabolites were determined with Testosterone-Immulite assay. HPLC separation of radiolabeled Eurasian lynx fecal extract indicated that the majority of testosterone metabolites are substances with a higher polarity than testosterone. Only minor proportion of radioactivity co-eluted with authentic testosterone and dihydrotestosterone. Enzymatic hydrolysis and solvolysis of the fecal extract were insufficient to liberate testosterone. After solvolysis relatively more activity was eluated the position of DHT, but the majority of metabolites remained unaffected. The EIA measured substantial amount of immunoreactivity, which corresponded with two radioactive peaks. Additionally, both immunoassays recognized two metabolites, which were only minor components according to their radioactivity. The Immulite assay was able to recognize a metabolite at the position of dihydrotestosterone. HPLC separation of Iberian lynx feces extracts revealed a similar metabolite pattern determined by EIA that were typical for Eurasian lynx fecal extracts. Simultaneous analyses of fecal samples with both testosterone assays provided comparative results for both lynx species (Eurasian lynx, r2=0.488; p<0.001; Iberian lynx, r2=0.85, p<0.0001). Thus, seasonal reproductive activity of male Eurasian lynx was demonstrated also by Immulite -assay, confirming high testosterone levels during breeding season in March/April as previously documented with EIA. Preliminary results on testosterone measurements in Iberian lynx feces confirmed the suitability of the applied Immulite test in this highly endangered species.

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters, morphology and function of testis and accessory glands.

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.